This project is designed to provide information on mechanism(s) of cell death induced by photodynamic therapy (PDT). The long-range goal is an understanding of the selective tumor eradication mediated by PDT. We have classified photosensitizing agents based on their sub-cellular targets: bcl-2, lysosomes, the plasma membrane, or combinations of these targets. We propose a hypothesis that relates PDT targets to death mechanisms: Sensitizers that target bcl-2 can initiate apoptosis by removing a barrier to interactions of bak/bax with the mitochondrial membrane. This process is mimicked by the bcl-2 antagonist HA 14-1. The binding of some sensitizers to bcl-2 is such that some anti-oxidants cannot protect the protein from photodamage. Sensitizers that target lysosomes cause the release of proteases leading to cleavage of the pro-apoptotic protein bid to t-bid. The latter promotes bax polymerization, resulting in an apoptotic response. Photosensitizers that target the plasma membrane can catalyze concomitant photodamage to caspases, likely a result of sensitizer relocalization during irradiation. Stress kinase activation is triggered by reactive oxygen species (ROS) and is pertinent only at low PDT doses that are insufficient to initiate the apoptotic program. At high PDT doses, these responses are downstream of caspase-3 activation and the commitment to death. Experiments have been designed to test the elements of this hypothesis, using a variety of murine and human cell lines, both adhering and in suspension culture. We also plan to explore the basis for the finding that PDT that targets bcl-2 spares the close analog bcl-xL. This implies differences in localization or sensitizer:protein affinity and suggests that bcl-xL alone cannot prevent PDT-induced apoptosis. A sub- contract with Dr. Kevin M Smith (Louisiana State University) will provide a supply of sensitizers including a limited synthetic effort directed at preparing agents able to target bcl-2 or bcl-xL. Collaborative arrangements with Drs. Reiners (co- investigator) and Kim (consultant) continue arrangements that have been productive during the previous interval of support.